This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PI will be developing multiphoton (MPM) optical imaging methods to systematically study human glioblastoma multiform spheroids embedded in various 3D ECMs. These experimental cancer model systems are used to mimic the in vivo gliomas. An MPM ratiometric redox fluorometry method based on intrinsic cellular fluorescence from reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) to study mitochondrial energy metabolism will be developed by the PI. Coupling these finding with the evaluations of mitochondrial membrane using a dye and visualizing F-actin distribution in conjunction with mitochondrial signals will furnish a better understanding on how ECM alters the growth, differentiation and metabolic states of the malignant glioma cells. The imaging methods developed will help cancer investigators to study key cancer properties such as cell survival and metastasis and can allow accurate measurements of tumor response to treatments. The studies proposed will benefit current therapies and prognosis for glioblastoma multiform.